The first objective of the proposed research is to understand the role of the Akt substrate and Rab GTPase- acfivafing protein (GAP) AS160 in regulafing the trafficking of the glucose transporter GLUT4 and in thus controlling glucose uptake in primary adipocytes. GLUT4 is responsible for the uptake of glucose into fat and muscle cells in the presence of insulin. ASI 60 is highly expressed in adipocytes, whereas its expression levels in skeletal muscles are low. Studies with adipocytes in culture have demonstrated that under basal condifions ASI60 uses its GAP domain to inhibit the Rabs responsible for controlling the movement of GLUT4-containing vesicles to the cell surface. In the presence of insulin ASI60 is phosphorylated and its GAP domain is inactivated. This leads to the acfivafion ofthe specific Rabs, translocafion of GLUT4 to the cell surface and increased glucose uptake. Preliminary studies with adipocytes from ASI 60 knockout (ASI60 KO) mice demonstrated age-dependent changes in glucose uptake;increased basal and normal glucose uptake in adipocytes from 6 week old mice, and normal basal, but impaired insulin-sfimulated glucose uptake in 3 month old mice. Under Aim 1 we will confirm the preliminary results on glucose uptake, and use cell surface biofinylafion and subcellular fractionafion to understand how the lack of ASI 60 affects GLUT4 subcellular distribufion under basal and insulin-sfimulated condifions in primary adipocytes. Our second objective is to determine if ASI 60 plays a role in regulafing fatty acid uptake in primary adipocytes. Fatty acid translocase (FAT/CD36) is responsible for fatty acid uptake in fat and muscle cells. Previous studies showed that the subcellular distribufion of FAT/CD36 is controlled by insulin in a similar fashion to GLUT4. However, the signaling pathways involved in the regulafion of FAT/CD36 distribufion have not been established. Mice lacking a funcfional gene for Tbddl, a close homolog of AS160 predominanfiy expressed in skeletal muscle, show impaired insulin-sfimulated glucose uptake in skeletal muscles similar to 3 month old ASI60 KO adipocytes. In addifion, increased fatty acid uptake and oxidafion was found in skeletal muscles ofthe Tbcldl mutant mice. Based on these findings we hypothesize that AS160 may play a role in the regulation of fatty acid uptake in adipocytes. We will test this hypothesis by measuring fatty acid uptake and determining the subcellular distribufion of FAT/CD36 in primary ASI 60 KO adipocytes using cell surface biofinylafions and subcellular fracfionations. Understanding how ASI 60 affects glucose and fatty acid uptake in adipocytes will be key to understanding molecular mechanisms leading to impaired energy homeostasis in type 2 diabetes.